The P granule antibody KT3 recognizes epitopes in both PGL-1 and PGL-3

The KT3 antibody is a commercially available antibody that recognizes the P granule protein PGL-3 (Takeda et al., 2008). Using immunostaining and western blotting of purified peptide fragments, we show that KT3 recognizes both PGL-3 and its paralog PGL-1 , likely through a shared epitope in the intrinsically disordered region.


A. PGL proteins
Diagram showing different domains in PGL-1, PGL-2 and PGL-3 and MUSCLE alignment of the PGL-1 and PGL-3 intrinsically disordered regions (not including the RGG repeats).Regions of homology between PGL-1 and PGL-3 are highlighted in red and may correspond to regions recognized by KT3.The PGL-2 IDR did not show significant stretches of homology and thus was excluded from this analysis.

C: Western analyses
Coomassie-stained gel (left panel) and western blot of same probed with KT3 antibody (right panel).Recombinant proteins were purified as fusions with maltose binding protein (MBP) and cleaved to release MBP and untagged PGL.Arrows point to bands recognized by the KT3 antibody.Bands of higher-than-expected molecular weight likely correspond to uncleaved MBP fusions.PGL-3 is full length, MBP is maltose binding protein.The next five lanes are PGL-3 segments as indicated (Refer to diagram A for amino acid coordinates).PGL-1 is full length PGL-1.

Description
The KT3 antibody and other antibodies obtained via antigen subtraction are widely used reagents for studying C. elegans (Takeda et al. 2008, Hanazawa et al. 2011, Schmidt et al., 2021).Antigen subtraction is a two round hybridoma production and screening method designed to raise monoclonal antibodies against random, relatively low abundance proteins present in a tissue lysate.The KT3 antibody was originally characterized (Takeda et al., 2008) as an antibody against PGL-3, a 75kD P granule protein (Kawasaki et al., 2004).In immunostaining experiments in embryos, KT3 recognizes P granules and an unidentified (pgl-3 independent) epitope in muscle (Takeda et al., 2008).
We conclude that KT3 recognizes P granule proteins PGL-1 and PGL-3, likely through a shared epitope in the IDR.

RNAi
L4 larvae were placed on IPTG-induced lawns of HT115 bacteria bearing L4440-based plasmid for 24 hours at 25°C and then shifted to 20°C for one hour to avoid temperature dependent changes on P granules.The RNAi clone for pgl-1 came from the genomic RNAi feeding library (Medical Research Council Gene Services, Source BioScience, Nottingham, UK; Kamath et al., 2003).

Immunostaining
For embryo immunostaining, adult worms were placed into M9 on poly-l-lysine (0.01%)-coated slides and pressed under a coverslip to extrude embryos.For adult germline immunostaining, adult worms were placed in M9 buffer with 10 mM levamisole before worms were sliced open to extrude germlines.For both embryos and adult germlines, slides were laid on aluminum blocks pre-chilled by dry ice for more than 5 min.Coverslips were promptly removed to freeze-crack and permeabilize embryos and germlines.Both were then incubated overnight in methanol at −20°C.Prior to antibody treatment, fixed germlines were treated with 4% paraformaldehyde for one hour.Slides were blocked in 0.1% PBS Tween with 0.1% BSA for 1 hour, followed by an overnight incubation with 200 μL of KT3 primary antibody (1:100, DSHB).The following day, samples were incubated with a secondary antibody at room temperature for two hours (Jackson ImmunoResearch Labs Cat# 115-605-068 1:500).They were then mounted with Vectashield Antifade Mounting Media with DAPI.

Western Blot
Recombinant proteins were diluted to 1 μM prior to denaturation and loading onto a 4-12% Bis-Tris pre-cast gels (Bio-Rad Hercules, CA).Western blot transfers onto PVDF membranes were run for 1 hour at 4°C.Membranes were blocked for 30 minutes in PBS with 5% milk.The KT3 antibody (DSHB) was diluted by 1:100 and incubated overnight.The secondary incubation occurred for two hours at room temperature (Thermofisher Scientific cat# 62-6720 1:4000, Jackson labs).